An internal domain of Exo70p is required for actin-independent localization and mediates assembly of specific exocyst components.
The exocyst consists of eight rod-shaped subunits that align in a side-by-side manner to tether secretory vesicles to the plasma membrane in preparation for fusion. Two subunits, Sec3p and Exo70p, localize to exocytic sites by an actin-independent pathway, whereas the other six ride on vesicles along actin cables. Here, we demonstrate that three of the four domains of Exo70p are essential for growth. The remaining domain, domain C, is not essential but when deleted, it leads to synthetic lethality with many secretory mutations, defects in exocyst assembly of exocyst components Sec5p and Sec6p, and loss of actin-independent localization. This is analogous to a deletion of the amino-terminal domain of Sec3p, which prevents an interaction with Cdc42p or Rho1p and blocks its actin-independent localization. The two mutations are synthetically lethal, even in the presence of high copy number suppressors that can bypass complete deletions of either single gene. Although domain C binds Rho3p, loss of the Exo70p-Rho3p interaction does not account for the synthetic lethal interactions or the exocyst assembly defects. The results suggest that either Exo70p or Sec3p must associate with the plasma membrane for the exocyst to function as a vesicle tether.
Hutagalung
Alex H
AH
Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA.
Coleman
Jeff
J
Pypaert
Marc
M
Novick
Peter J
PJ
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055571:Secretory Pathway / Q000502:physiology; D033921:Vesicular Transport Proteins; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
D000199:Actins; C103132:EXO70 protein, S cerevisiae; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C103131:SEC5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; C074647:RHO3 protein, S cerevisiae; D020741:rho GTP-Binding Proteins